The present invention relates to novel tumor antigen peptide derivatives.
It is known that the immune system, particularly T cells, plays an important role in vivo in tumor rejection. Indeed, infiltration of lymphocytes having cytotoxic effects on tumor cells has been observed in human tumor foci (Arch. Surg., 126:200-205, 1990), and cytotoxic T lymphocytes (CTLs) recognizing autologous tumor cells have been isolated from melanomas without great difficulties (e.g., Immunol. Today, 8:385, 1987; J. Immunol., 138:989, 1987; and Int. J. Cancer, 52:52-59, 1992). In addition, the results of clinical treatment of melanomas by T cell introduction also suggest the importance of T cells in tumor rejection (J. Natl. Cancer. Inst., 86:1159, 1994).
Although it has long been unknown about target molecules for CTLs attacking autologous tumor cells, the recent advance in immunology and molecular biology has gradually revealed such target molecules. Specifically, it has been found that using T cell receptors (TCRs), CTL recognizes a complex between a peptide, called tumor antigen peptide, and a major histocompatibility complex class I antigen (MHC class I antigen, and in the case of human, referred to as HLA antigen), and thereby attacks autologous tumor cells.
Tumor antigen peptides are generated from proteins specific for tumors, that is, tumor antigen proteins. Thus, the proteins are intracellularly synthesized and then degraded in cytoplasm into the peptides by proteasome. On the other hand, MHC class I antigens (HLA antigens) formed at endoplasmic reticulum, when bind to the above tumor antigen peptides, are transported via cis Golgi to trans Golgi, i.e., the mature side and carried to the cell surface where they are presented as an antigen. A tumor-specific CTL recognizes this complex presented as an antigen, and exhibits its anti-tumor effects through the cytotoxic effect or production of lymphokines (Rinsho-Menneki, 27(9):1034-1042, 1995). As a consequence of such elucidation of a series of actions, it has become possible to treat tumors by using tumor antigen proteins or tumor antigen peptides as so-called cancer vaccines which enhance tumor-specific CTLs in a patient.
As such tumor antigen proteins, T. Boon et al. identified a protein named MAGE from human melanoma cells for the first time in 1991 (Science, 254:1643-1647, 1991), and thereafter several additional tumor antigen proteins have been identified from melanoma cells.
As reviewed by T. Boon et al. (J. Exp. Med., 183, 725-729, 1996), tumor antigen proteins hitherto identified can be divided into the following four categories.
Tumor antigen proteins belonging to the first category are those which are expressed in testis only as normal tissues, while they are expressed in melanoma, head and neck cancer, non-small cell lung cancer, bladder cancer and others, as tumor tissues. Among tumor antigen proteins in this category are the above-described MAGE and analogous proteins constituting a family of more than 12 members (J. Exp. Med., 178:489-495, 1993), as well as BAGE (Immunity, 2:167-175, 1995) and GAGE (J. Exp. Med., 182:689-698, 1995), all of which have been identified in melanoma cells.
Although some of such tumor antigen proteins in this category are highly expressed in melanoma, the expression thereof is observed in only 10 to 30% of patients having a particular tumor other than melanoma, and therefore, they cannot be applied widely to treatments or diagnoses of various tumors.
Tumor antigen proteins belonging to the second category are those which are expressed only in melanocytes and retina among normal tissues, and in melanomas among tumor tissues. Since these tissue-specific proteins are highly expressed in melanomas, they would function as tumor antigen proteins specific for melanomas. Among tumor antigen proteins in this category are tyrosinase (J. Exp. Med., 178:489-495, 1993), MART-1 (Proc. Natl. Acad. Sci. USA, 91:3515, 1994), gp100 (J. Exp. Med., 179:1005-1009, 1994), and gp75 (J. Exp. Med., 181:799-804, 1995). Genes encoding these proteins have all been cloned from melanoma cells. Melan-A (J. Exp. Med., 180:35, 1994), which has been separately isolated, has proved to be identical with MART-1.
However, the tumor antigen proteins of this category cannot be used widely in the treatments or diagnoses of various tumors, since they are not expressed in tumors other than melanoma.
Tumor antigen proteins belonging to the third category are those which are expressed as tumor antigen peptides recognized by CTL as a result of tumor-specific mutations. Among tumor antigen proteins in this category are mutated CDK4 (Science, 269:1281-1284, 1995), xcex2-catenin (J. Exp. Med., 183:1185-1192, 1996), and MUM-1 (Proc. Natl. Acad. Sci. USA, 92:7976-7980, 1995). In CDK4 and xcex2-catenin, a single amino acid mutation increases the binding affinity of the peptides to MHC class I antigen, which allows them to be recognized by T cells. In MUM-1, an intron, which normally is not translated, is translated due to mutation, and the resultant peptide is recognized by T cells. However, since such mutations occur at low frequency, they cannot be applied widely to treatments or diagnoses of various tumors.
Tumor antigen proteins belonging to the fourth category are IDS those widely expressed in normal tissues and also recognized by CTL, example of which includes P15 identified from melanoma cells (J. Immunol. 154:5944-5955, 1995).
Tumor antigen proteins or peptides hitherto known have been identified in the following manners.
A set of tumor cell and CTL attacking the tumor cell (usually established from lymphocytes of the same patient from whom the tumor cells are obtained) is first provided. Then, the set of cells are used to directly identify tumor antigen peptides, or to determine a gene encoding tumor antigen protein, from which the corresponding tumor antigen peptide is identified.
In the method where tumor antigen peptides are directly identified, tumor antigen peptides bound to MHC class I antigens in tumor cells are extracted under acidic conditions, and separated into various peptides using high-performance liquid chromatography. Tumor antigen peptides are then identified by pulsing cells expressing MHC class I antigen but not expressing tumor antigen protein (for example, B cells from the same patient) with the resultant peptides and examining the reactivity with CTL. The sequences of the peptides thus identified are then determined by, for example, mass spectrometry. In this way, tumor antigen peptides derived from Pmel 17 which is the same molecule as gp100 have been identified from melanoma cells (Science, 264:716-719, 1994).
In the method where a gene encoding tumor antigen protein is first obtained which is followed by the identification of the corresponding tumor antigen peptide, such a gene can be cloned using molecular biological techniques. MHC class I antigen gene and cDNAs prepared from tumor cells are co-transfected into cells not expressing tumor antigen proteins (for example, COS cells) for transient expression. The expression products are then repeatedly screened on the basis of their reactivity with CTL to isolate a gene encoding tumor antigen protein. In this way, the genes encoding the above-mentioned MAGE, tyrosinase, MART-1, gp100, and gp75 have been cloned.
The following method can be used to actually deduce and identify a tumor antigen peptide bound to and presented with MHC class I antigen (HLA antigens) on the basis of information about tumor antigen gene. Fragments of various sizes are first prepared from a gene encoding tumor antigen protein by means of PCR, exonucleases, or restriction enzymes, or the like, and cotransfected with MHC class I antigen gene into cells not expressing tumor antigen proteins (e.g., COS cells) for transient expression. The region(s) which include tumor antigen peptides are then identified on the basis of their reactivity with CTL. Subsequently, peptides are synthesized on the basis of the identified regions. Cells expressing MHC class I antigen but not expressing tumor antigen proteins are then pulsed with the synthesized peptides to identify the tumor antigen peptides, for example, by examining their reactions with CTL (J. Exp. Med., 176:1453, 1992; J. Exp. Med., 179:24, 759, 1994). The sequence regularities (motifs) for peptides, which are bound and presented by certain types of MHC such as HLA-A1, -A0201, -A0205, -A11, -A24, -A31, -A6801, -B7, -B8, -B2705, -B37, -Cw0401, and -Cw0602 have been known (Immunogenetics, 41:178-228, 1995), and therefore, candidates for tumor antigen peptides may also be designed by making reference to such motifs, synthesized and examined in the same way as described above (Eur. J. Immunol., 24:759, 1994; J. Exp. Med., 180:347, 1994).
According to procedures as described above, various tumor a antigen proteins and tumor antigen peptides have been hitherto identified. As described above, however, some of the known tumor antigen proteins are expressed only in limited tumors, and others are expressed only in a small number of patients having a particular tumor even if they are expressed in various kinds of tumor, and therefore, they cannot be used widely in the treatments or diagnoses of various tumors.
One of the purposes of the present invention is to provide tumor antigen peptide derivatives which can be used widely and universally without limitations regarding the kind of tumor or the subjects, in particular, tumor antigen proteins, tumor antigen peptides corresponding thereto, and derivatives thereof which can be widely applied to treatments and diagnoses of tumors with high incidence such as squamous cell carcinoma. Thus, it is one of purposes of the present invention to provide novel tumor antigen peptide derivatives derived from a tumor other than melanomas, in particular, from a squamous cell carcinoma, and also methods, compositions, kits, and the like, for treating, preventing or diagnosing tumors by the use of said tumor antigen peptide derivatives. It is also a purpose of the present invention to provide tumor antigen peptide derivatives restricted to HLA-A24, which is an HLA antigen carried by a large part of human subjects.
To this end, the present inventors have established a squamous cell carcinoma cell line KE-4 derived from esophageal cancer (hereinafter referred to as esophageal cancer cell line KE-4 or simply as KE-4), and also established CTL (hereinafter referred to as KE-4CTL) which recognizes tumor antigen peptides restricted to HLA antigens such as HLA-A2601, HLA-A2402, and the like, which are expressed by said KE-4 (Cancer Res., 55:4249-4253, 1995).
Fibroblast cell line VA-13 was then cotransfected with a recombinant plasmid of cDNA library prepared from KE-4 and a recombinant plasmid containing HLA-A2601 cDNA. The screening of a gene(s) encoding novel tumor antigen protein was carried out by treating the resulting transfectants with KE-4CTL, and measuring the amount of produced IFN-xcex3 to determine whether KE-4CTL was activated or not. As a result, the inventors succeeded in cloning a novel gene encoding a novel tumor antigen protein for the first time from tumor cells other than melanomas. The nucleotide sequence of the cloned gene is shown in SEQ ID NO: 1 and the deduced amino acid sequence is shown in SEQ ID NO: 2.
Subsequently, the present inventors tried to identify the portions in the amino acid sequence of the above tumor antigen protein that actually function as tumor antigen peptides, and identified various tumor antigen peptide portions restricted to HLA-A26, HLA-A24, and the like.
Among them, a peptide having the amino acid sequence at positions 690 to 698 (SEQ ID NO: 3) in the amino acid sequence shown in SEQ ID NO: 2 was identified as an HLA-A24-restricted tumor antigen peptide. The present inventors then prepared various peptide derivatives by altering an amino acid residue(s) in the HLA-A24-restricted tumor antigen peptide shown in SEQ ID NO: 3 and determined their activities, which revealed that the derivatives also have the activity as a tumor antigen peptide.
The present invention was completed on the basis of such findings.
Thus, the gist of the present invention is to provide tumor is antigen peptide derivatives which comprise all or part of an amino acid sequence wherein one to several amino acid residues in the amino acid sequence shown in SEQ ID NO: 3 are altered, and which derivatives are capable of binding to HLA-A24 antigen and thus being recognized by cytotoxic T cells.